1. Field of the Invention
This invention resides in the field of materials and reagents for tests of human blood.
2. Description of the Prior Art
Tests for determining blood coagulation rates are useful in diagnosing bleeding abnormalities and for monitoring the blood coagulation behavior of a patient that is undergoing treatment or medication for the prevention of blood clot formation. Blood coagulation is caused by the formation of fibrin which results from the action of thrombin on fibrinogen, a soluble component of normal blood, by a succession of reactions that involve a series of blood clotting factors. Thrombin itself is formed from prothrombin by two primary pathways, an extrinsic pathway and an intrinsic pathway, and different coagulation tests measure the viability of one or both of these pathways. The viability of the extrinsic pathway is measured by a determination known in the art as the prothrombin time (PT), while the viability of the intrinsic pathway is measured by a determination known in the art as the activated partial thromboplastin time (APTT). By their own methodologies, both PT and APTT each serve as an indication of the length of time needed for the blood clotting to occur.
Prothrombin time (PT) tests provide an indication of the presence and activity of prothrombin, otherwise known as Factor II, as well as four other clotting factors—Factors I, V, VII, and X. When the level or activity of one or more of these factors is abnormally low, or the activity is blocked by abnormal substances in the subject's blood, the PT value (expressed in seconds) is high. In some cases, this is an indication of a disease condition, while in others the high value is an indication of a successful therapy. Certain medical conditions, for example, are treated by the administration of medications such as heparin and warfarin, that purposely prevent or retard the formation of blood clots. The PT value for a subject undergoing warfarin medication, for example, will be about 1.5 to 2.5 times the result obtained on a healthy subject. The PT value for a healthy subject not under such medication typically falls within the range of about 10 to about 13 seconds.
An activated partial thromboplastin time (APTT) test is commonly performed prior to surgery to confirm that the subject has normal blood clotting behavior. Like PT, APTT is also used to monitor the administration of blood-thinning medications such as heparin, typically by performing the test every two hours and correcting the dosage of the medication until an optimal dosage is reached. For subjects with normal clotting behavior, the APTT value will be within the range of about 25 to about 39 seconds.
A variety of analyzers and reagents are presently available to clinical laboratories for both PT and APTT determinations from commercial suppliers. One such supplier is Diagnostica Stago, Inc. of Parsippany, N.J., USA, whose products include STA-PPT[A]® reagent for APTT tests and STA®-Neoplastine CI Plus reagent for PT tests. The STA-PPT[A]® reagent is used in a test that involves recalcification of plasma in the presence of a standardized amount of cephalin (used as a platelet substitute) and a particulate activator. The STA-Neoplastine CI Plus reagent is used in combination with calcium thromboplastin. An additional supplier is HemoSense, Inc., San Jose, Calif., USA, whose INRatio® Meter is a test device that measures PT and the International Normalized Ratio (INR) which is the ratio of the patient PT to the mean normal PT for a population. The INRatio® Meter obtains these values from one drop of fresh capillary blood from a fingerstick, by use of a recombinant human thromboplastin reagent, and determines the change in impedance of the sample upon the conversion of fibrinogen to fibrin. Another test device is the i-STAT® analyzer sold by i-STAT Corporation of East Windsor, N.J., USA. The i-STAT® analyzer is a hand-held device that contains an artificial thrombin substrate that contains a linkage resembling the site on fibrinogen that thrombin normally cleaves to form a fibrin clot. Thrombin in the blood sample causes cleavage of the substrate and the resulting release of an electroactive compound that is detected amperometrically. The i-STAT analyzer can also be used to measure activated clotting time (ACT), which is the time required for complete activation of the coagulation cascade. ACT determinations are useful for monitoring moderate- and high-level heparin therapy through analysis of arterial and venous blood samples. Complete activation is indicated when extensive or localized clots form as the result of the conversion of fibrinogen to fibrin in the presence of activated thrombin.
Compositions serving as blood sample substitutes are routinely used in conjunction with these various tests, as standards, references, and controls. These compositions are useful in monitoring the precision and accuracy of the instruments or devices, monitoring the condition of any reagents used with the instruments or devices, and comparing patient samples with other samples or with fixed values. Sample substitutes are also used for training purposes when introducing new users to a particular device, instrument, or procedure. A goal in formulating a sample substitute (referred to herein for convenience as a control) is to achieve a composition that is as sensitive as an actual patient sample to all of the analytical variances that are likely to be encountered, and one that reads a value that is within the range of the medical decision point of the assay. The optimal composition is also one that is stable for hours or days after preparation or reconstitution. Other desirable features are low cost, ease of manufacturing, and reproducibility from one lot to the next.
One control that is currently available is the Stago STA-Coag Control (Catalog No. 00679 of Diagnostica Stago), a bi-level lyophilized control that contains citrated normal and abnormal human plasma to represent positive and negative levels, respectively. A tri-level control sold under the name LYPHOCHEK® Coagulation Control is available from Bio-Rad Laboratories, Inc., Hercules, Calif. USA (Catalog Nos. 744, 745, and 746), prepared from processed human plasma and preservatives. Since neither the Stago STA-Coag Control and the LYPHOCHEK® Coagulation Control contain erythrocyte materials, neither of these controls is a whole blood coagulation control. This is a disadvantage since the optimal control material for any whole blood coagulation test, particularly those designed for point-of-care testing, is one that is similar in constitution to the actual sample being tested, and by lacking erythrocytes and erythrocyte components, plasma-based controls lack a major class of components that are present in samples derived from whole blood. Formulations for stable whole blood coagulation controls are disclosed by Speck, R. E., et al. (Analytical Control Systems, Inc.), U.S. Pat. No. 5,939,325, issued Aug. 17, 1999. The Speck controls include non-primate-derived coagulation factors in combination with primate-derived coagulation factors to compensate for any loss of activity over time of the more labile primate-derived factors.